Method and kit for detection of autoimmune chronic urticaria

ABSTRACT

Disclosed is a rapid, non-invasive and highly specific and sensitive diagnostic assay for the identification of individuals with autoimmune chronic urticaria, which makes use of CD203c, and in some embodiments, additional proteins, as a marker for the disease. Test kits for diagnosis of an individual suspected of having autoimmune chronic urticaria are also disclosed. Also disclosed are a method of identifying compounds useful for treating autoimmune chronic urticaria and a method of treating autoimmune chronic urticaria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)from U.S. Provisional Application No. 60/664,090, filed Mar. 21, 2005.The entire disclosure of U.S. Provisional Application No. 60/664,090 isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention generally relates to a method and assay kit for thediagnosis of autoimmune chronic urticaria, and a method to treatautoimmune chronic urticaria.

BACKGROUND OF THE INVENTION

Chronic idiopathic urticaria (CU) is defined as recurrent hivesoccurring for at least 6 weeks. In the majority of cases, there is noidentifiable trigger, despite extensive evaluation for an underlyingetiology. A subset of these patients is classified as having autoimmuneurticaria defined by the presence of a functional IgG antibody to thealpha-subunit of the high-affinity IgE receptor (FcεRIα) or to IgE(Kaplan, 2004). These antibodies trigger mast cell and basophildegranulation by the engagement of this receptor. Functional IgGantibody to the receptor has been identified in approximately 30-40% ofpatients with CU, and anti-IgE antibody has been identified in another5-10% of patients. Non-functional antibodies to FcεRI may be found inother autoimmune conditions (Fiebiger, 1998).

Techniques to detect the autoantibody to FcεRIα include Western blot andenzyme linked immunoabsorbant assay (ELISA), which are technically timeconsuming and fail to identify antibodies with histamine releasingproperties. Detection methods for functional antibodies include theautologous serum skin test (ASST) and basophil histamine release (BHR).The ASST involves an intradermal injection of the patient's serum intothe skin with observation for a wheal and flare reaction. ASST isapproximately 70% sensitive and 80% specific when compared with thebasophil histamine release (BHR) assay (Aabroe, 1999).

Recently, flow cytometry has been used to identify activated basophilsin both allergic disease and chronic urticaria. Upregulation of threeproteins (CD63, CD203c, and CD69) on peripheral blood basophils frompatients with CU has been described (Vasgar, 2003), although nocorrelation with autoimmune CU or histamine release was described forany of these markers.

With regard to CD63, two studies have been published demonstrating thatsera of patients with CU and positive ASST induce higher expression ofCD63 when compared to skin test negative chronic urticaria sera (Wedi,2000; Gyimsi, 2004). CD63, a member of the transmembrane-4 superfamily,is a basophil and mast cell activation marker that is expressed as aresult of the fusion between intracytoplasmic granules and the plasmamembrane. It has been proposed that it rapidly appears on the basophilsurface upon the addition of anti-IgE, allergen or IL-3 (Knol, 1991;Ebo, 2004). However, CD63 is not specific to basophils and mast cells,and can be expressed on other cells present in the peripheral blood,i.e., monocytes and platelets (Buhring, 2004).

Specifically, Wedi et al. (Wedi, 2000), who used interleukin-3 (IL-3) toactivate cells in whole blood, found increased CD63 expression in 70% ofASST positive CU patients, in 45% of ASST negative CU patients, and in35% of controls. Wedi et al. was not able to demonstrate specificity ofthe assay for patients with CU and the functional positive ASST, andcould not correlate CD63 expression with histamine releasing activity,nor could Wedi et al. show that CD63 expression was characteristic ofpatients with ASST+ sera or with CU patients in general (noting the highpercentage of negative controls with increased CD63 expression). Gyimesiet al (Gyimesi, 2004,) attempted to improve upon the results of Wedi etal. by using donor basophils from highly sensitized atopic individualsas the test cells. Gyimesi et al. found elevated CD63 expression inducedby 11/12 ASST positive sera (92%) and 3/18 (17%) of ASST negative sera.However, the use of the highly sensitized atopic donor cells appears tobe a requirement for the improve sensitivity of this test.

Therefore, there remains a need in the art for a rapid, in vitro assaythat is minimally invasive, and is capable of distinguishing autoimmunepatients from patients with other forms of urticaria, including otherforms of chronic urticaria, such as that caused by allergy (atopicpatients).

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a method to diagnoseautoimmune chronic urticaria. The method includes the steps of: (a)contacting isolated cells with a test biological sample from anindividual being diagnosed for autoimmune chronic urticaria, wherein theisolated cells are capable of expressing CD203c and FcεRIα, andupregulate the expression of CD203c upon stimulation of the FcεRIα; and(b) detecting expression of CD203c expression on the cells. Upregulationof CD203c on the cells in the presence of the test biological sample, ascompared to CD203c expression on the cells in the absence of contactwith the biological sample, or as compared to CD203c expression on thecells when contacted with a negative control sample from an individualwho does not have autoimmune chronic urticaria, indicates that theindividual has autoimmune chronic urticaria.

In one aspect of this embodiment, the test biological sample is a fluidsample, including, but not limited to, serum or whole blood from theindividual. In one aspect, the isolated cells are provided as a wholeblood sample from a donor or as isolated peripheral blood cells from adonor. In one aspect, the isolated cells are provided as substantiallypurified cells from a donor. In another aspect, the isolated cells areprovided as a cell line. In yet another aspect, the isolated cells arefrom a donor that does not have autoimmune chronic urticaria. In oneaspect, the isolated cells have been selected as having highupregulation of CD203c expression after stimulation with a granulocyteactivating agent and/or after stimulation of FcεRIα on the cells, ascompared to isolated cells from a population of normal individuals thatdo not have autoimmune chronic urticaria. In one aspect, the cells arefrom a non-atopic donor. In another aspect, the isolated cells are notcultured with interleukin-3 prior to the step of contacting. In yetanother aspect, the isolated cells are selected from: basophils and mastcells.

In one aspect of this embodiment of the invention, the step of detectingis performed by flow cytometry. In one aspect, the step of detecting isperformed using a CD203c antibody or antigen-binding fragment thereof,including, but not limited to, a monoclonal antibody.

In one aspect of this embodiment of the invention, the cells expressCD13, and the method further comprises detecting expression of CD13 bythe cells. Increased expression of CD13 in the presence of the testbiological sample, as compared to expression of CD13 by the cells in theabsence of contact with the biological sample, or as compared to CD13expression on the cells when contacted with a negative control samplefrom an individual who does not have autoimmune chronic urticaria,further indicates that the individual has autoimmune chronic urticaria.

In another aspect of this embodiment of the invention, the cells expressCD164, and the method further comprises detecting expression of CD164 bythe cells. Increased expression of CD164 in the presence of the testbiological sample, as compared to expression of CD164 by the cells inthe absence of contact with the biological sample, or as compared toCD164 expression on the cells when contacted with a negative controlsample from an individual who does not have autoimmune chronicurticaria, further indicates that the individual has autoimmune chronicurticaria.

In yet another aspect of this embodiment of the invention, the cellsexpress CD63, and the method further comprises detecting CD63 expressionon the cells. Increased expression of CD63 in the presence of the testbiological sample, as compared to CD63 expression on the cells in theabsence of contact with the biological sample, or as compared to CD63expression on the cells when contacted with a negative control samplefrom an individual who does not have autoimmune chronic urticaria,further indicates that the individual has autoimmune chronic urticaria.

In another aspect of this embodiment of the invention, the methodadditionally includes testing the individual by histamine release assay,wherein a positive histamine release assay further indicates that theindividual has autoimmune chronic urticaria.

In yet another aspect of this embodiment of the invention, the methodadditionally includes testing the individual by autologous serum skintesting (ASST), wherein a positive ASST test further indicates that theindividual has autoimmune chronic urticaria.

Another embodiment of the present invention relates to a kit for thediagnosis of autoimmune chronic urticaria. The kit includes: (a)isolated cells that are capable of expressing CD203c and FcεRI and thatupregulate the expression of CD203c upon stimulation of FcεRI; and (b) areagent for the detection of CD203c expression on the cells. In oneaspect, the reagent for detection of CD203c expression on the cells is aCD203c antibody or antigen-binding fragment thereof, including, but notlimited to, a monoclonal antibody. In one aspect, the kit also includesa reagent selected from a positive control reagent and a negativecontrol reagent. A positive control reagent can include, but is notlimited to, fMLP and/or anti-FcεRIα. A negative control reagent caninclude, but is not limited to, a buffer. In one aspect of thisembodiment, the isolated cells are basophils.

Yet another embodiment of the present invention relates to a method toidentify a compound that inhibits autoimmune chronic urticaria. Themethod includes the steps of: (a) contacting isolated cells that arecapable of expressing CD203c and FcεRIα, and that upregulate theexpression of CD203c upon stimulation of the FcεRIα, with an agent thatstimulates the expression of CD203c by the cells; (b) contacting theisolated cells with a putative regulatory compound; and (c) detectingexpression of CD203c expression on the cells. Prevention or inhibitionof upregulation of CD203c on the cells in the presence of the putativeregulatory compound, as compared to the upregulation of CD203cexpression on the cells in the absence of contact with the putativeregulatory compound, indicates that the putative regulatory compound isa candidate for inhibition of autoimmune chronic urticaria.

Another embodiment of the present invention relates to a method to treatautoimmune chronic urticaria, comprising administering to the individuala pharmaceutical composition that inhibits the expression or biologicalactivity of CD203c.

BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION

FIGS. 1A-1C show basophil identification and expression of CD203c. FIG.1A shows a side scatter and CD45—gating region (boxed area) containedthe basophils located between the monocytes and lymphocytes. FIG. 1Bshows the gated cells that were selected by high IgE and high CD203cexpressing cells. FIG. 1C is a histogram of CD203c expression afteraddition of buffer (shaded area) and after addition of serum from achronic urticaria patient with a positive ASST (M1 defines a regionwhere ≦1% of the basophils express CD203c after incubation with buffer).

FIG. 2 shows the mean percent (±SEM) CD203c expression above baseline bysera from normal controls (N=11), disease controls (N=4), all CUpatients (N=32), and positive controls (i.e., fMLP and anti-FcεRI).

FIG. 3 shows the percent CD203c expression by normal individuals, CUpatients with negative ASST, and CU patients with positive ASST(horizontal bars represent means).

FIG. 4 shows the correlation of CD203c expression with % total histaminerelease.

FIG. 5 shows the correlation of percent CD203c expression and ASST meanwheal diameter.

DETIALED DESCSRIPTION OF THE INVENTION

This invention generally relates to methods, reagents and kits that areuseful for the detection of autoimmune chronic urticaria. The inventioncan be used to identify patients that have autoimmune chronic urticaria(e.g., a diagnostic, prognostic or monitoring method) and to furtherunderstand the mechanisms of chronic urticaria for the development oftherapeutic strategies directed at preventing or treating this disease.In addition, the present invention generally relates to theidentification of a therapeutic target for the treatment of chronicurticaria, namely, CD203c, a cell surface protein expressed primarily bybasophils, and to therapeutic strategies for chronic urticaria. Thepresent invention also relates to the use of other markers that followthe kinetics of CD203c expression in basophils as additional oralternative markers to CD203c, including, but not limited to, CD13 andCD164.

Approximately 40% of patients with chronic urticaria (CU) haveantibodies to the high affinity IgE receptor (FcεRI). Patients withauto-antibodies are currently identified by histamine release assay(HR), autologous serum skin testing (ASST) and Western blot. CD203c isexpressed specifically on basophils, mast cells and their CD34+progenitor cells and is upregulated by cross-linking of FcεRI receptors.The present inventors have shown that CD203c is a highly useful markerto identify autoimmune urticaria patients with functional antibodies tothe FcεRI receptor.

More particularly, the present inventors compared the effect of serafrom ASST+ (autoimmune) and ASST-chronic urticaria (CU) patients onbasophil CD203c expression to normal control sera, and determinedwhether expression of CD203c correlated with basophil histamine release(BHR). In addition, IgG depletion of sera from ASST positive individualswas performed to determine if the mechanism of CD203c upregulation isIgG-mediated.

The inventors have discovered that the sera from patients with CUupregulate basophil CD203c expression. Sera from patients with positiveASST (a known indicator of autoimmune chronic urticaria) inducedsignificantly higher CD203c expression compared to the sera frompatients with negative ASST and from normal controls. Specifically,while 20% of patient sera with negative ASSTs induced ≧5% basophilactivation compared to buffer control, whereas 77% percent of patientswith positive ASSTs had ≧5% basophil activation. Although sera from ASSTnegative patients induced higher CD203c expression than normal controls,the CD203c expression in these patients was nonetheless significantlylower than CD203c expression in ASST positive patients.

The sensitivity, specificity, positive predictive value and negativepredictive value of the method or detection of CD203c as a marker for CUwere calculated using the BHR assay as the gold standard to identifyfunctional antibodies to the FcεRIα receptor, and using >5% CD203cexpression as the cut-off value for a positive test. Using thesecriteria, CD203c expression demonstrated a sensitivity of 77%,specificity of 82%, a positive predictive value of 83% and a negativepredictive value of 75%. Therefore, detection of CD203c correlates wellwith results from the current standard for detection of autoimmune CU.

In addition, the present inventors have demonstrated that the mechanismof expression of CD203c in CU patients is mediated by IgG. CD203c isknown to be upregulated by cross-linking of FcεRIα by transport ofpreformed CD203c molecules to the surface (14). The decline in CD203cexpression following IgG depletion of representative sera is consistentwith an IgG-mediated mechanism (see Example 4), which can include an IgGantibody against the FcεRIα subunit or against IgE.

The method of the present invention is particularly useful because itcan identify patients that have functional FcεRI autoantibodies in arapid, in vitro assay that is minimally invasive and is capable ofdistinguishing autoimmune patients from patients with other forms ofurticaria, including other forms of chronic urticaria, such as thatcaused by allergy (atopic patients). The method is more sensitive thandiagnosis using a different marker, such as CD63, because while CD63 canbe detected on a variety of cell types, CD203c is a much more specificmarker of basophils and basophil degranulation, and therefore, is a muchmore specific marker of the cell types and mechanisms associated withthe development of autoimmune chronic urticaria. Furthermore, incontrast to assays described for the detection of CD63 (Wedi, 2000;Gyimesi, 2004), the present invention does not require the use ofinterleukin-3 to stimulate test cells, nor the use of cells from atopicpatients to increase sensitivity. Moreover, prior to the presentinvention, the use of CD203c as a diagnostic marker for autoimmunechronic urticaria had not been described.

CD203c belongs to the type II transmembrane protein family and is amultifunctional ecto-enzyme called ectonucleotide pyrophosphatasephophodiesterase 3 (E-NPP3) that catalyzes the cleavage of a number ofmolecules including deoxynucleotides and nucleotide sugars. It alsocontains a somatomedin B-like domain and a cell adhesive motif, buttheir potential functions are unknown. Among leukocytes, CD203c appearsto be selectively expressed on the basophil/mastocytes lineage. Prior tothe present invention, CD203c had been used to evaluate IgE mediatedhypersensitivity to latex and aeroallergens (Boumiza, 2002; Hauswirth,2002). For example, Boumiza et al. compared basophil activation testsusing either CD63 or CD203c in the diagnosis of latex allergy and foundthat the sensitivity was considerably higher with CD203c (75% comparedto 50% with CD63) (Boumiza, 2002), likely due to superior recognition ofbasophils using CD203c, and the higher level of expression of CD203c inactivated basophils as compared to CD63. Without being bound by theory,the present inventors propose that the difference in response betweenCD203c and CD63 can also be explained by their different mechanism ofupregulation. For example, PGD2 is a strong inducer of CD203cexpression. However, it does not stimulate CD63 expression (15). Thekinetics of upregulation of CD203c is fast and peaks after 5-15 minutes,whereas that of CD63 is slow and peaks after 20-40 min (Hennersdorf,2005). CD203c is expressed on both resting and activated basophils andis upregulated in response to cross-linking of the FcεRIα receptor(Binder, 2002).

Some recently identified surface markers follow the upregulationkinetics of either CD203c or CD63 (CD13, CD164 and CD107a). Theupregulation of CD13 and CD164 is fast and similar to that of CD203c andaccordingly, these two proteins represent additional markers that can beused as an alternative to or in addition to the use of CD203c toidentify patients with autoimmune chronic urticaria as described herein.On the other hand, the surface increase of CD107a is slow and followsthe kinetics of CD63 (Monneret, 2005).

Accordingly, one embodiment of the present invention relates to a methodto diagnose autoimmune chronic urticaria in a patient. The methodincludes a first step of contacting isolated cells that express CD203cand FcεRI (e.g., mast cells or basophils, and most preferably,basophils) with a test biological sample from a patient being diagnosedfor autoimmune chronic urticaria. The isolated cells upregulate CD203cexpression after stimulation either via the FcεRI and/or by a differenttype of stimulation that is sufficient to upregulate CD203c (e.g., witha granulocyte activating reagent, such as fMLP). The method furtherincludes a second step of detecting expression of CD203c expression onthe cells. Upregulation of CD203c on the cells in the presence of thetest biological sample, as compared to CD203c expression on the cells inthe absence of contact with the biological sample, and/or as compared toCD203c expression on the cells when contacted with a normal controlsample, indicates that the patient has autoimmune chronic urticaria. Inother embodiments of this invention, CD13 or CD164 may be substitutedfor CD203c. In another embodiment, the method can include detection ofany one or more of CD13, CD164 and CD63, in addition to the detection ofCD203c, which may be performed simultaneously with the detection ofCD203c or sequentially. In additional embodiments, the method caninclude detection of functional auto-antibodies by histamine releaseusing a basophil histamine release (BHR) assay and/or an autologousserum skin test (ASST).

Chronic urticaria (CU) is a disease defined by a period of whealing(welts, hives) lasting for weeks to many years. Urticaria is a vascularreaction, usually transient, involving the upper dermis, representinglocalized edema caused by dilatation and increased permeability of thecapillaries, and marked by the development of wheals. The lesions areusually pruritic. The causes of chronic urticaria can include,infectious disease, malignancy, allergic reactions, pseudoallergicreactions, and autoimmunity. Autoimmune chronic urticaria is the subjectof the present invention, and refers to chronic urticaria that isassociated with the presence in the individual of autoantibodies,particularly autoantibodies against the high affinity IgE receptor(FcεRI), although anti-IgE antibodies may also contribute to thedisease. These autoantibodies are believed to cross-link the a subunitsof FcεRI on mast cells or basophils, resulting in the degranulation ofthe cells and release of histamine. Although anti-FcεRI are found inpatients with a variety of autoimmune disorders, in autoimmune chronicurticaria, the antibodies are identified as “functional” for causing thedegranulation of basophils and mast cells, as well as histamine release.Functional anti-FcεRI antibodies are typically identified by autologousserum skin testing (ASST) and may be confirmed by histamine release (HR)assays, immunoblotting, or ELISA. The present invention provides a rapidand sensitive assay that has been demonstrated to correlate inspecificity and sensitivity with these known tests for functionalanti-FcεRI antibodies, but is less invasive and time consuming thanthese assays.

The first step in the method of the present invention includes a step ofcontacting isolated cells that express the marker of interest (i.e.,CD203c and/or in some embodiments, CD13, CD164 and/or CD63) and FcεRIαwith a test biological sample from a patient being diagnosed forautoimmune chronic urticaria. In general, any methods or techniquesdescribed herein for CD203c can be applied to other markers to be usedin the alternative, or in addition to, the CD203c marker of theinvention. The cells to be used in the assay of the present inventioncan be any cells that express the marker of interest (i.e., CD203c andin some embodiments, CD13, CD164 and/or CD63) and FcεRIα, or thatexpress functional portions of the marker of interest and/or the FcεRIα,and that upregulate the expression of the marker (e.g., CD203c) upon stimulation of FcεRIα, such as by cross-linking of FcεRIα. Such cells canbe identified by exposing a candidate cell to anti-FcεRI (andparticularly anti-FcεRIα) and/or to another reagent that stimulates theactivation of the cell (e.g., a granulocyte stimulator, such as fMLP),whereby such exposure results in upregulation of expression of themarker (e.g., CD203c).

In one aspect of the invention, the cells used in the assay are mastcells or basophils, with basophils being particularly preferred. Oneadvantage of the present invention is that the cells, and particularlybasophils, can be provided in the form of whole blood or a fractionthereof from a selected donor (described below), because the assay,being based on the detection of a marker that is selective for basophilsand mast cells (e.g., CD203c), will specifically detect basophil andbasophil degranulation (e.g., specifically related to autoimmune chronicurticaria) without the need to separate the basophils from otherperipheral blood cells. However, the present invention also contemplatesthe provision of suitable cells in any form, including in whole blood,in a fraction thereof (e.g., peripheral blood cells isolated therefrom),as a partially or substantially purified sample of basophils (or mastcells or other suitable cells), or as a cell line (e.g., isolated,cloned, immortalized, genetically modified, etc.).

In one aspect of the invention, the cells can be isolated from a singledonor or from multiple donors, as long as the cells upregulateexpression of CD203c (or another desired marker) upon stimulation ofFcεRIα. Preferably, the cells have been selected to upregulateexpression of CD203c upon stimulation: of FcεRIα and, if the cell is abasophil, upon stimulation of FcεRIα and/or a granulocyte stimulator,such as FMLP. Particularly preferred cells to use in an assay of theinvention are cells meeting the above criteria that are upregulateCD203c to a significant degree upon stimulation of FcεRI so that theresult of testing in the assay can be clearly distinguished abovebackground CD203c expression. In particular, cells can be selected fromamong a pool of possible cells (e.g., cells from different donors can bescreened) that upregulate CD203c in response to stimulation of FcεRI(and perhaps also using another stimulator, such as FMLP) to astatistically more significant degree than the other cell samples in thepool. The skilled practitioner will readily be able to select a suitabledonor. Preferably, the donor from which the cells used in the assay areobtained or derived does not have autoimmune chronic urticaria, and morepreferably, does not have any form of urticaria. In one embodiment, thedonor is non-atopic, although atopic donors may also be used.

An example of a method to select a suitable donor cell is described inthe Examples section. For example, whole blood from a panel of potentialdonors can be screened for sufficient upregulation of CD203c onbasophils (CD203c/IgE positive cells) in response to a known stimulatorof CD203c (e.g., fMLP) and a known stimulator of FcεRIα (e.g.,anti-FcεRIα antibody or another agent that engages or cross-links thereceptor). Preferably, the expression of CD203c is upregulated on thecell in response to a known stimulator of CD203c at least 5%, and morepreferably, at least 30%, and even more preferably, at least 50% (or anypercentage in between, in whole number increments—at least 5%, at least6%, etc.), as compared to the expression of CD203c in the absence of theknown stimulus of CD203c (e.g., fMLP) or as compared to the normal(negative or background) control. Alternatively, the expression ofCD203c is upregulated on the cell in response to a known stimulator ofCD203c at least 1.5 fold, and more preferably, at least 3 fold, and evenmore preferably, at least 6 fold, as compared to the expression ofCD203c in the absence of the known stimulus of CD203c (e.g., fLP) or ascompared to the normal (negative or background) control. These criteriafor CD203c expression can also be used to identify other cells that areuseful in the present invention (e.g., cell lines). These criteria canalso be used with other markers described herein (e.g., CD13, CD164and/or CD63) to identify cells useful for screening for these markers,although identifying a cell based on CD203c expression is believed to besufficient to screen for these other markers, if desired.

The cells selected for use in the present invention can be obtainedrepeatedly from a given donor source(s), immortalized or otherwiseprepared as cell lines that can be repeatably cultured, maintained,frozen, rethawed, and/or stored, and then used in the method of theinvention. One may also use a publicly available cell line that meetsthe criteria of upregulating CD203c upon stimulation as describedherein. For example, a basophil cell lines derived from a patient withmyelogenous leukemia are available through the American Type CultureCollection (ATCC®)) as ATCC Deposit Nos. CRL-2099™, CRL-2100™ andCRL-2101™.

As discussed above, CD203c belongs to the type II transmembrane proteinfamily and is a multifunctional ecto-enzyme called ectonucleotidepyrophosphatase phophodiesterase 3 (E-NPP3) that catalyzes the cleavageof a number of molecules including deoxynucleotides and nucleotidesugars. It also contains a somatomedin B-like domain and a cell adhesivemotif, and among leukocytes, is selectively expressed on thebasophil/mastocytes lineage. The nucleic acid and amino acid sequencesfor human CD203c are known in the art (e.g., see Accession No.gi:4826896 (amino acid sequence) and Accession No. gi:25453478 (nucleicacid sequence), both of which are incorporated herein by reference intheir entireties).

CD13 (also known as human aminopeptidase-N) is a zinc-bindingaminopeptidase-N enzyme expressed on the surface of early committedprogenitors of granulocytes and monocytes (CFU-GM) and by all cells ofthese lineages as they mature (including on activated basophils), onendothelial cells, epithelial cells from renal proximal tubules andintestinal brush border, bone marrow stromal cells, osteoclasts, andcells lining bile duct canaliculi, and on a small proportion of largegranular lymphocytes (Soderberg et al 1993) but not other lymphocytes.The nucleic acid and amino acid sequences for human CD13 are known inthe art (e.g., see Accession No. gi:4502095 (amino acid sequence) andAccession No. gi:4502094 (nucleic acid sequence), both of which areincorporated herein by reference in their entireties).

CD164 (also known as sialomucin) is a mucin-like receptor, orsialomucin, expressed by human CD34⁺ hematopoietic progenitor cells andis expressed by activated basophils. Its functions include mediating, orregulating, haematopoietic progenitor cell adhesion and the negativeregulation of their growth and/or-differentiation. The nucleic acid andamino acid sequences for human CD164 are known in the art (e.g., seeAccession No. gi:21361274 (amino acid sequence) and Accession No.gi:34222157 (nucleic acid sequence), both of which are incorporatedherein by reference in their entireties).

CD63 is a member of the transmerbrane-4 superfamily, and is expressed onmany cells in the peripheral blood, including on activated basophils andmast cells as a result of the fusion between intracytoplasmic granulesand the plasma membrane. The nucleic acid and amino acid sequences forhuman CD63 are known in the art (e.g., see Accession No. gi:4502679(amino acid sequence) and Accession No. gi:34328936 (nucleic acidsequence), both of which are incorporated herein by reference in theirentireties).

Human FcεRIα is the high affinity receptor for immunoglobulins of theIgE isotype. The amino acid and nucleic acid sequences encoding thecomponents of this receptor are known in the art, and thethree-dimensional structure of FcΕRIα has been solved (e.g., see Garmanet al., 1998, Cell 95(7):951-961 (structure denoted IF2Q); Garman etal., 2000, Nature 406(6793):259-266 (structure denoted IF6A); and Garmanet al., 2001, J. Mol. Biol., 311(5):1049-62).

The conditions under which a cell that expresses CD203c (or anothermarker described herein) is contacted with a patient test sample, suchas by mixing, are conditions in which the expression of CD203c is notstimulated (activated) or upregulated if essentially no test sample ispresent. For example, such conditions include normal culture conditionsin the absence of a known activating compound or other equivalentstimulus of expression of CD203c, and in the case of basophils, ofbasophil degranulation. The patient test sample is then contacted withthe cells. Effective culture conditions include, but are not limited to,appropriate media, temperature, pH and oxygen conditions that permit thegrowth of the cells used in the test. An appropriate, or effective,medium is typically a medium comprising growth factors and assimilablecarbon, nitrogen and phosphate sources, as well as appropriate salts,minerals, metals and other nutrients, such as vitamins. Culturing iscarried out at a temperature, pH and oxygen content appropriate for thecell. Such culturing conditions are within the expertise of one ofordinary skill in the art.

A patient sample most typically includes any bodily fluid (although cellor tissue samples are not excluded) that may contain functionalautoantibodies against FcεRIα (i.e., an anti-FcεRI that upregulatesCD203c and causes degranulation of granulocytes, and particularly,basophils). More specifically, according to the present invention, theterm “test sample” or “patient sample” or “test biological sample” canbe used generally to refer to a sample of any type which contains, ormay contain, the antibodies to be evaluated by the present method,including but not limited to, a sample of isolated cells, a tissuesample and/or a bodily fluid sample. According to the present invention,a sample of isolated cells is a specimen of cells, typically insuspension or separated from connective tissue which may have connectedthe cells within a tissue in vivo, which have been collected from anorgan, tissue or fluid by any suitable method which results in thecollection of a suitable number of cells for evaluation by the method ofthe present invention. A tissue sample, although similar to a sample ofisolated cells, is defined herein as a section of an organ or tissue ofthe body which typically includes several cell types and/or cytoskeletalstructure which holds the cells together. One of skill in the art willappreciate that the term “tissue sample” may be used, in some instances,interchangeably with a “cell sample”, although it is preferably used todesignate a more complex structure than a cell sample. A tissue samplecan be obtained by a biopsy, for example, including by cutting, slicing,or a punch. A bodily fluid sample, like the tissue sample, contains thecells to be evaluated for marker expression or biological activityand/or may contain a soluble biomarker that is secreted by cells, and isa fluid obtained by any method suitable for the particular bodily fluidto be sampled. Bodily fluids suitable for sampling include, but are notlimited to, blood, mucous, seminal fluid, saliva, breast milk, bile andurine. The present invention will most typically use a whole blood orserum sample from a patient, and most preferably uses a serum sample.

The period of contact of the cells with the patient sample being testedcan be varied, and can be determined by one of skill in the art. Forexample, the period of contact can be minutes or hours, and is typicallyperformed in from about 5 to 60 minutes. As used herein, the term“contact period” refers to the time period during which cells are incontact with the patient sample being tested. The term “incubationperiod” refers to the entire time during which cells are allowed to growor bind to an agent prior to evaluation, and can be inclusive of thecontact period. Thus, the incubation period includes all of the contactperiod and may include a further time period during which the patientsample being tested is not present but during which expression of CD203cis allowed to continue or is stopped prior to scoring.

According to the present invention, the method includes the step ofdetecting the expression of CD203c (or another marker) in the cells.This step can include determining whether CD203c expression isupregulated as compared to a control, downregulated as compared to acontrol, or unchanged as compared to a control. Therefore, the step ofdetecting expression does not require that expression of CD203c actuallyis upregulated, but rather, can also include detecting that theexpression of CD203c has not changed (i.e., detecting no upregulation ofexpression of CD203c or detecting non-significant upregulation ofexpression of CD203c).

Methods to measure CD203c expression or to detect expression levels ofany protein marker generally include, but are not limited to: flowcytometry, fluorescence activated cell sorting (FACS), enzyme-linkedimmunosorbant assay (ELISA), radioimmunoassay (RIA),immunoprecipitation, surface plasmon resonance, chemiluminescence,fluorescent polarization, phosphorescence, immunohistochemical analysis,matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)mass spectrometry, and/or microcytometry, microscopy. In one aspect ofthe invention, CD203c expression is detected using flow cytometry. Inone aspect, two or more markers are simultaneously detected (e.g.,CD203c and CD63; CD203c and CD13 and/or CD164, etc.).

In one embodiment, the CD203c (or other marker) is detected using anantibody or antigen-binding fragment thereof that selectively binds tothe CD203c (or other marker). Antibodies that selectively bind to CD203ccan be produced using CD203c protein information available in the art.More specifically, the phrase “selectively binds” refers to the specificbinding of one protein to another (e.g., an antibody, fragment thereof,or binding partner to an antigen), wherein the level of binding, asmeasured by any standard assay (e.g., an immunoassay), is statisticallysignificantly higher than the background control for the assay. Forexample, when performing an immunoassay, controls typically include areaction well/tube that contain antibody or antigen binding fragmentalone (i.e., in the absence of antigen), wherein an amount of reactivity(e.g., non-specific binding to the well) by the antibody or antigenbinding fragment thereof in the absence of the antigen is considered tobe background. Binding can be measured using a variety of methodsstandard in the art including enzyme immunoassays (e.g., ELISA),immunoblot assays, etc.). In one embodiment, antibodies that bind to anepitope (linear or conformational) of the long form, but not the shortform, of FBLN-3 are envisioned. Such antibodies typically bind to anepitope that comprises at least a portion of the extracellular domain ofCD203c.

Antibodies useful in the test kit and methods of the present inventioncan include polyclonal and monoclonal antibodies, divalent andmonovalent antibodies, bi- or multi-specific antibodies, serumcontaining such antibodies, antibodies that have been purified tovarying degrees, and any functional equivalents of whole antibodies.Isolated antibodies of the present invention can include serumcontaining such antibodies, or antibodies that have been purified tovarying degrees. Whole antibodies of the present invention can bepolyclonal or monoclonal. Alternatively, functional equivalents of wholeantibodies, such as antigen binding fragments in which one or moreantibody domains are truncated or absent (e.g., Fv, Fab, Fab′, or F(ab)₂fragments), as well as genetically-engineered antibodies or antigenbinding fragments thereof, including single chain antibodies orantibodies that can bind to more than one epitope (e.g., bi-specificantibodies), or antibodies that can bind to one or more differentantigens (e.g., bi- or multi-specific antibodies), may also be employedin the invention.

The invention also extends to non-antibody polypeptides, sometimesreferred to as antigen binding partners or antigen binding peptides,that have been designed to bind selectively to the protein of interest(e.g., CD203c). Examples of the design of such polypeptides, whichpossess a prescribed ligand specificity are given in Beste et al. (Proc.Natl. Acad Sci. 96:1898-1903, 1999), incorporated herein by reference inits entirety.

Methods of detecting CD203c (or another marker) expression may alsoinclude detecting transcription of the marker in the donor cells. Such amethod can include, but is not limited to, extraction of cellular mRNAand Northern blotting using labeled probes that hybridize to transcriptsencoding all or part of a gene encoding the marker (e.g., CD203c);amplification of mRNA using marker-specific primers, polymerase chainreaction (PCR), and reverse transcriptase-polymerase chain reaction(RT-PCR), followed by quantitative detection of the product by any of avariety of means; extraction of total RNA from the cells, which is thenlabeled and used to probe cDNAs or oligonucleotides encoding all or partof a gene encoding CD203c; in situ hybridization; and detection of areporter gene. Other methods of detecting transcription are known in theart and can be applied here.

According to the present invention, a probe or primer is a nucleic acidmolecule which typically ranges in size from about 8 nucleotides toseveral hundred nucleotides in length. Such a molecule is typically usedto identify a target nucleic acid sequence in a sample by hybridizing tosuch target nucleic acid sequence under stringent hybridizationconditions. As used herein, stringent hybridization conditions refer tostandard hybridization conditions under which nucleic acid molecules areused to identify similar nucleic acid molecules. Such standardconditions are disclosed, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989.Sambrook et al., ibid., is incorporated by reference herein in itsentirety (see specifically, pages 9.31-9.62). In addition, formulae tocalculate the appropriate hybridization and wash conditions to achievehybridization permitting varying degrees of mismatch of nucleotides aredisclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138,267-284; Meinkoth et al., ibid., is incorporated by reference herein inits entirety.

Agents useful for detecting expression of a marker of the presentinvention can be conjugated to a detectable tag or detectable label.Such a tag can be any suitable tag which allows for detection of themarker using the selected technique (described above) and includes, butis not limited to, any composition or label detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include biotinfor staining with labeled streptavidin conjugate, magnetic beads (e.g.,Dynabeads™), fluorescent dyes (e.g., fluorescein, texas red, rhodamine,green fluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I,³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and colorimetriclabels such as colloidal gold or colored glass or plastic (e.g.,polystyrene, polypropylene, latex, etc.) beads.

According to the present invention, a “control level” of CD203cexpression (or another marker) is a baseline level, and in someembodiments (but not all embodiments, depending on the method), a normalor negative level, of CD203c expression against which a test level ofexpression (i.e., in the test sample) can be compared. Therefore, it canbe determined, based on the control or baseline level of CD203cexpression, whether a sample to be evaluated causes a measurableincrease or substantially no change in CD203c expression in the cellsincluded in the test, as compared to the negative baseline level. In oneaspect, the baseline level is a negative or normal control level that isindicative of the CD203c expression expected from contact with a normal(i.e., healthy, negative control, non-autoimmune chronic urticaria)biological sample. Therefore, the term “negative control” used inreference to a baseline level of CD203c expression typically refers to abaseline level established from a sample from a population ofindividuals which is believed to be normal (i.e., healthy, not sufferingfrom autoimmune CU). It is noted that exposure of the cells to the“negative control” results in a lower level of CD203c expression thanwould be detected from exposure to a sample from a patient withautoimmune CU.

In another embodiment, a “positive control” baseline is used (includingin addition to the negative control), which refers to a level of CD203cexpression established by exposing the cells in the assay to a knownstimulator of FcεRI cross-linking and/or a known stimulator of CD203cexpression (e.g., anti-FcεRI and fMLP), or to a sample from a patientthat is known to have autoimmune CU. It is noted that this “positivecontrol” will most typically induce have a higher level of CD203cexpression than in a normal or negative control sample.

It will be appreciated by those of skill in the art that a baseline neednot be established for each assay as the assay is performed but rather,a baseline can be established by referring to a form of storedinformation regarding a previously determined baseline level of CD203cexpression for a given control sample with the same test cells. Such aform of stored information can include, for example, but is not limitedto, a reference chart, listing or electronic file of population orindividual data regarding “normal” (negative control) or positive CD203cexpression, or any other source of data regarding baseline CD203cexpression that is useful for the patient to be diagnosed.

A positive diagnosis indicates that the patient has, is developing, oris at risk of developing, autoimmune chronic urticaria. In order toestablish a positive diagnosis, the level of CD203c expression isincreased over the established baseline by an amount that isstatistically significant (i.e., with at least a 95% confidence level,or p<0.05). The values obtained from the test and/or any control samplesare statistically processed using any suitable method of statisticalanalysis to establish a suitable baseline level using methods standardin the art for establishing such values. Preferably, detection of atleast about a 5-10% change in CD203c expression in the sample ascompared to the baseline level results in a positive diagnosis. Morepreferably, detection of at least about a 30% change in CD203cexpression from the sample as compared to the baseline level results ina positive diagnosis. More preferably, detection of at least about a 50%change, and more preferably at least about a 70% change, and morepreferably at least about a 90% change, or any percentage change between5% and higher in 1% increments (i.e., 5%, 6%, 7%, 8% . . . ) in CD203cexpression induced by the sample as compared to the baseline levelresults in a positive diagnosis of autoimmune chronic urticaria for thetest sample, and therefore, for the patient. In one embodiment, a 1.5fold change in CD203c expression induced by the sample as compared tothe baseline level results in a positive diagnosis. More preferably,detection of at least about a 3 fold change, and more preferably atleast about a 6 fold change, and even more preferably, at least about a12 fold change, and even more preferably, at least about a 24 foldchange, or higher, or any fold change from 1.5 and higher in incrementsof 0.5 fold (i.e., 1.5, 2.0, 2.5, 3.0 . . . ) in CD203c expression ascompared to the baseline level, results in a positive diagnosis ofautoimmune chronic urticaria. These criteria can also be used toevaluate other markers described herein (e.g., CD13, CD164, CD63).

In one embodiment of the method of the present invention, as discussedabove, in addition to evaluating the expression of CD203c in response tocontact with an individual's test biological sample, one canadditionally or alternatively evaluate a change in expression of CD13and/or CD164 in the test cells in response to contact with theindividual's test biological sample. Detection of an increase inexpression of either CD13 or CD164 in cells after contact with the testbiological sample, as compared to CD13 or CD164 expression,respectively, on the cells in the absence of contact with the biologicalsample, or as compared to CD13 or CD164 expression, respectively, on thecells when contacted with a negative control sample from an individualwho does not have autoimmune chronic urticaria, indicates that thepatient has autoimmune chronic urticaria. If used together with CD203cdetection, these markers can be detected at the same time as CD203c(e.g., by multiple labeling using flow cytometry) or in sequentialsteps.

In another embodiment of the method of the present invention, inaddition to evaluating the expression of CD203c (and/or CD13 and/orCDD164) in response to contact with an individual's test biologicalsample, one can evaluate any one or more of: CD63 expression by the testcell in response to contact with the biological sample; histaminerelease in response to contact with the biological sample, or autologousserum skin test reactions using the biological sample. Methods of CD63detection are as described herein for CD203c, or can be performed asdescribed previously (Wedi, 2000; Gyimesi, 2004). The histamine releaseassay (HR or BHR) is performed as described in the Examples or aspreviously described (Ferrer, 1999; Platz er et al., 2005, “Validationof basophil histamine release against the autologous serum skin test andoutcome of serum-induced basophil histamine release studies in a largepopulation of chronic urticaria patients”, Allergy 60:1152-6). Anautologous serum skin test (ASST) is performed as described in theExamples or as previously described (Platzer et al., supra; Sabroe,1999). Such additional evaluations are used to confirm, or furthervalidate, the results of the method of the present invention, but arenot required to practice the method of the present invention.

Another embodiment of the invention relates to test or assay kitscontaining the reagents necessary to evaluate a patient for autoimmunechronic urticaria using the method of the invention. Such reagents willbe apparent to those of skill in the art given the present disclosure.For example, such a test kit may include, (a) cells (e.g., basophils)for use in the assay (e.g., that upregulate CD203c expression afterstimulation with a granulocyte activating reagent and/or afterstimulation of FcεRIα on the cells); and (b) a reagent for the detectionof CD203c expression on the cells (e.g., either by detection of proteinexpression or detection of transcription of RNA encoding CD203c). Forexample, the reagent for detection of CD203c expression on the cells caninclude, but is not limited to, a CD203c antibody or antigen-bindingfragment thereof, such as a monoclonal antibody, or probes or primersuseful for the detection of CD203RNA. The kit may further include otheruseful reagents, such as a means for detecting the binding of theantibody to CD203c (e.g., a secondary antibody, a label, an enzyme,etc.). The kit may further include positive control reagents (e.g., fMLPand anti-FcεRIα) and/or a negative control reagent (e.g., a buffer). Thekit may include reagents for detecting other markers described herein(e.g., CD13, CD164, CD63), or reagents useful for performing a BHR(e.g., reagents for the detection of histamine) or ASST.

The kit preferably contains any means of detecting the expression oractivity of a CD203c, and preferably includes a probe; PCR primers; oran antibody, antigen binding peptide, or fragment thereof, that binds toCD203c or a gene encoding CD203c. The kit can include any reagent neededto perform a diagnostic method envisioned herein. The antibody, orfragment thereof, can be conjugated to another unit, for example amarker or immobilized to a solid carrier (subs trate). The kit can alsocontain a second antibody for the detection of CD203c:antibodycomplexes. In one embodiment, the kit can contain a reagent fordetecting a control marker characteristic of a given cell type (e.g., anantibody or antigen binding fragment thereof). The antibody or fragmentthereof, or a probe or primer, may be present in free form orimmobilized to a substrate such as a plastic dish, a test tube, a testrod and so on. The kit can also include suitable reagents for thedetection of and/or for the labeling of positive or negative controls,wash solutions, dilution buffers and the like, as well as instructionsfor using the assay kit.

Another embodiment of the invention relates to the use of CD203c as atarget for the development of therapeutic compositions and to strategiesand methods for the prevention and/or treatment of autoimmune chronicurticaria. Such a method includes, for example, administering to thepatient a pharmaceutical composition that inhibits the binding ofautoantibodies to FcεRIα and/or inhibits the activation of the basophilor mast cell, and particularly the degranulation of such cells, whichcan be monitored by evaluating the expression of CD203c. Such compounds,by inhibiting expression of CD203c directly or indirectly can be usefulfor treating or preventing autoimmune chronic urticaria. Such compoundscan include, but are not limited to, antibodies and antigen-bindingfragments thereof, small molecules, proteins and peptides, RNAi,aptamers, ribozymes and antisense nucleic acid molecules.

RNA interference (RNAi) is a process whereby double stranded RNA, and inmammalian systems, short interfering RNA (siRNA) or short hairpin RNA(shRNA), is used to inhibit or silence expression of complementarygenes. In the target cell, siRNA are unwound and associate with an RNAinduced silencing complex (RISC), which is then guided to the mRNAsequences that are complementary to the siRNA, whereby the RISC cleavesthe mRNA. shRNA is transfected into a target cell in a vector where itis transcribed, and then processed by DICER enzymes to form siRNA-likemolecules that activate RISC, which, as with siRNA, is then guided tothe mRNA sequences that are complementary to the shRNA, whereby the RISCcleaves the mRNA.

Aptamers are short strands of synthetic nucleic acids (usually RNA butalso DNA) selected from randomized combinatorial nucleic acid librariesby virtue of their ability to bind to a predetermined specific targetmolecule with high affinity and specificity. Aptamers assume a definedthree-dimensional structure and are capable of discriminating betweencompounds with very small differences in structure.

A ribozyme is an RNA segment that is able to perform biologicalcatalysis (e.g., by breaking or forming covalent bonds). Morespecifically, ribozymes are antisense RNA molecules that function bybinding to the target RNA moiety and inactivate it by cleaving thephosphodiester backbone at a specific cutting site.

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

One may use CD203c (or CD13 or CD164) as a target marker for theidentification of therapeutic compounds that inhibit or preventdegranulation and histamine release by cells that are typically targetedby the autoantibodies of autoimmune CU patients (e.g., basophils). Asused herein, the term “putative” or “candidate”, when used with regardto a regulatory compound, refers to compounds having an unknownregulatory activity, at least with respect to the ability of suchcompounds to regulate expression of a gene or protein or the biologicalactivity of a protein as described herein. In the method of identifyinga regulatory compound according to the present invention, the method canbe a cell-based assay, or non-cell-based assay.

For example, this method can be performed in a cell-based assay usingsubstantially the same method as the diagnostic assay, except that thecells are stimulated by a known stimulator of CD203c (e.g., fMLP andanti-FcεRI, or a sample from a patient known to have autoimmune chronicurticaria), instead of with a test biological sample. The cells are alsocontacted with a putative regulatory compound, either before stimulationof the cells (to determine the ability of the compound to preventupregulation of expression of the CD203c) or simultaneously with orafter stimulation of the cells (to determine the ability of the compoundto reduce/inhibit/downregulate upregulation of expression of CD203c).The assay then includes a step of detecting whether the putativeregulatory compound prevents or reduces (or downregulates) theupregulation of CD203c on the cells in response to the known stimulus.In this method, CD203c serves as a marker to indicate that thecharacteristics of activation of the test cell are inhibited orprevented by the putative regulatory compound. Since the presentinventors have shown that upregulation of CD203c on a basophilcorrelates with histamine release, this assay is believed to be areasonable indicator of compounds that can inhibit basophil activationand/or histamine release by the basophil (or mast cell). Compounds thatprevent or reduce the upregulation of expression of CD203c by a cell areidentified as compounds that are candidates for inhibiting(ameliorating, reducing the symptoms or recurrence of) autoimmunechronic urticaria in a patient. Such compounds can then be furthervalidated using other assays (e.g., using other markers or testsdescribed herein) and in clinical trials.

One may also identify compounds that bind to CD203c (e.g., such as by animmunoassay or other binding assay), or bind to a nucleic acid moleculeencoding 203c (e.g., RNAi, ribozymes, antisense, or inhibitors oftranscription or translation of CD203c RNA or protein), or regulate theact ivity of CD203c (e.g., regulates the enzymatic activity of theprotein) using a non-cell based assay. Such assays will be apparent tothose of skill in the art given the target marker.

Compounds identified using such methods of the invention can be used toreduce or prevent the symptoms of autoimmune chronic urticaria in anindividual. Such compounds can include proteins, polypeptides, riucleicacid molecules, antibodies and binding fragments thereof, and smallmolecules (e.g., products of drug design or screening).

According to the present invention, the methods of the present inventionare suitable for use in an individual or with the cells or biologicalsample of an individual that is a member of the Vertebrate class,Mammalia, including, without limitation, primates, livestock anddomestic pets (e.g., a companion animal). Most typically, an individualwill be a human individual. The term “individual” can be interchangedwith the term “subject” or “patient” and refers to the subject of aprotocol or method according to the invention. Accordingly, anindividual can include a healthy, normal (non-diseased) individual, aswell as an individual who has or is at risk of developing autoimmunechronic urticaria or a symptom or indicator thereof as described herein.

Various aspects of the invention are described in the following example;however, the following example is provided for the purpose ofillustration and are not intended to limit the scope of the presentinvention.

EXAMPLES

The following Materials and Methods were used in Examples 1-4 below.

Patients and Controls

Chronic urticaria (CU) was defined as recurrent wheals occurring atleast 3 times per week for more than 6 weeks without an identifiablecause. Patients were excluded if they had evidence of a known triggeror, cause, including physical urticaria, urticarial vasculitis orallergic cause of their hives. Sera were obtained from thirty-twopatients with CU at the time of ASST and stored at −80° C.Antihistamines were held for at least 48 hours prior to collection ofsera. Eleven sera from healthy adult individuals were used as normalcontrols. Four sera with a high titer ANA ≧1:360, 3 with a history ofactive connective tissue disease, and the other with eosinophilicgastroenteritis, were used for disease controls. The study was approvedby the Institutional Review Board at the National Jewish Medical andResearch Center.

ASST

The test was performed by injecting 0.05 cc of the patient's own serumintradermally into the volar aspect of the forearm. Sterile saline andhistamine were used as negative and positive controls, respectively.Wheal and flare reactions were measured at 30 minutes. A mean whealdiameter ≧2 mm than the control was considered to be a positive ASST.

Basophil Donor

Whole blood from seven normal individuals was screened and one donor wasidentified based on the marked upregulation of CD203c on basophils(CD203c/IgE positive cells) in response to FMLP and anti-FcεRIαantibody. The basophil donor was atopic and had a serum IgE level of 154kU/L. The basophil donor provided informed consent and was bled ≦1 timeper week.

IgG Depletion

Selected patients and normal control patients sera were depleted of IgGusing a protein G-Sepharose column (Sigma, St. Louis, Mo.). Pre and postIgG depletion levels were measured by nephelometry (ImmageImmunochemistry System, Beckman Coulter, Fullerton, Calif.). All serahad post depletion levels of <33.3 mg/dL (the lowest level of detectionby nephelometry). Dilution of sera passing through the column wasaccounted for by measuring serum albumin levels pre and post depletion.

Antibodies

The following monoclonal antibodies were used: phycoerythrin(PE)-conjugated anti-human CD203c (Beckman Coulter, Fullerton, Calif.),peridinin chlorophyll protein complex (PerCP)-conjugated anti-human CD45(BD Bioscience, San Jose, Calif.), and flourescein isothiocyanate(FITC)-conjugated anti-human IgE (Caltag, Burlingame, Calif.).

Measurement of CD203c Surface Expression

The test was carried out on heparinized blood within 3-4 hours afterdrawing the donor's blood. Two hundred μL aliquots of the donor'sheparinized whole blood were incubated for 10 minutes at 37° C. with 40μL of sera from CU patients, normal controls, or IgG depleted sera. Forcontrols, 40 μL basophil stimulation buffer (20 mM HEPES, 125 mM NaCl, 5mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 0.5 mM glucose, and 0.1% BSA, pH 7.4)was added to 200 μL of donor's blood and used as the negative controlwhile 40 μL of −2 μM f-Met-Leu-Phe (fMLP, Sigma, St. Louis, Mo.) and 40μL of a 1:50 dilution of anti-FcεRIα receptor antibody (Upstate,Charlottesville, Va.) in Ca++ and Mg++ free phosphate buffered saline(PBS) were used as positive controls. The reactions were stopped byplacing the tubes on ice. Cells were then stained with PE-anti-humanCD203c, PerCP-anti-human CD45, and FITC-anti-human IgE at 4° C. in thedark for 30 minutes. Red cells were lysed with FACS Lysing Solution(Becton Dickinson, San Jose, Calif.). The cells were then washed oncewith 2 ml of PBS and fixed in 1% formaldehyde. The cells were thenanalyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose,Calif.).

Flow Cytometry

Basophils from whole blood were identified by the presence of CD203c onhigh expressing IgE positive cells (FIGS. 1A and 1B). The data areexpressed as the mean fluorescence intensity or the percentage of CD203cexpression. Percent CD203c expression is defined as the percent ofbasophils expressing more CD203c than ≧99% of basophils incubated withbuffer only. This was determined by defining an M1 region on thehistogram analysis (FIG. 1C).

Basophil Histamine Release (BHR)

Whole blood from the same donor for the CD203c assay was collected in aheparinized tube and BHR carried out according to the manufacturer'sinstructions (Beckman Coulter, Fullerton, Calif.). For the BHR wholeblood was diluted 1:7 in the histamine release buffer. Fifty μL ofstimuli including patients sera, anti-FcεRI (as a positive control), orhistamine release buffer (as a negative control) was added to 100 μL ofblood and incubated at 37° C. for 30 minutes. To determine totalhistamine, whole blood was diluted 1:20 in distilled water followed bytwo rapid freeze thaw cycles. Histamine concentrations in thesupernatants of centrifuged blood were performed by ELISA (BeckmanCoulter, Fullerton, Calif.) according to the manufacturer'sinstructions. Spontaneous histamine release was determined by the amountof histamine released by cells incubated with the histamine releasebuffer. Spontaneous release was <5% of total release. Positive histaminerelease was defined as >15% of total release (Ferrer, 1999).

Antinuclear Antibody Determination

Anti-nuclear antibodies (AN As) were detected by indirectimmunoflluorescence with HEp-2 cells (Bio-Rad Laboratories, Redmond,Wash.).

Statistics

Significant differences between groups were analyzed using a two-tailedunpaired t-test. Correlation was determined using Pearson Product Momentcorrelation. Sensitivity, specificity, negative and positive predictivevalue were also calculated.

Example 1

The following example demonstrates that CD203c expression on basophilsfrom chronic urticaria patients is elevated.

CD203c expression was higher in basophils incubated with sera from CUpatients compared to normal control individuals (FIG. 2). The meanpercent basophil CD203c expression of 11 normal control individuals was−0.6%±0.1 (±SEM) versus 20%±4 in 32 CU patients (p=0.002). Sera frompatients with high-titer ANAs (disease controls) did not induce basophilCD203c expression. The mean values of the positive controls fMLP andanti-FcεRIα were 34.5%±14.6 and 41.9%±9.4 (n=4), respectively.

CD203c expression was significantly higher after incubation of basophilswith sera from ASST positive CU patients than normal control sera(p<0.001) (FIG. 3). The mean percent CD203c expression was 30%±5 (SEM)(n=22) in basophils incubated with positive ASST positive CU sera versus−0.6%±0.1 for normal control sera (n=11). The range of percent CD203cexpression in cells incubated with ASST positive serum was −0.1% to63.3%. Seventeen of twenty-two ASST positive sera (77%) resulted inbasophil activation of greater than 5%. The range of percent basophilactivation in cells incubated with normal control serum was −1.2% to0.0% compared to buffer control. CD203c expression was alsosignificantly higher in basophils incubated with ASST positive sera thanwith ASST negative sera (p=0.006). The mean percent CD203c expressionwas 30%±5 versus 6%±3 respectively. The range of CD203c expression forbasophils incubated with ASST negative sera was 0.1% to 24%. Eight of 10ASST negative sera resulted in CD203c expression of ≦5%, and the othertwo resulted in 22% and 24% expression. Incubation with ASST negativesera also resulted in significantly higher CD203c expression thancontrol sera (p=0.04). Two patients with negative autologous serum skintests (ASST) and sera that induced an elevation of CD203c of over 5% mayrepresent those patients with false-negative ASST. However uponreanalyzing the data without the two outlying sera, ASST negativechronic urticaria sera still induced CD203c expression above controls.This finding indicates the presence of other undefined serum factorspresent in CU sera that can upregulate CD203c.

Example 2

The following example demonstrates that CD203c expression in autoimmuneCU patients correlates with basophil histamine release.

BHR was performed in 10 patients with negative ASSTs and in 18 patientswith positive ASST. All sera were not analyzed due to insufficientquantity. In patients with negative ASST, 1 of 10 (10%) had a positiveBHR, and 4 of 18 (22%) of patients with positive ASSTs had a negativeBHR. The percent CD203c expression correlated with the percent histaminerelease (R=0.6, p=0.001) (FIG. 4). The sensitivity, specificity,positive predictive value and negative predictive value were calculatedusing the BHR assay as the gold standard to identify functionalantibodies to the FcεRIα receptor, and using >5% CD203c expression asthe cut-off value for a positive test. Using these criteria, CD203cexpression demonstrated a sensitivity of 77%, specificity of 82%, apositive predictive value of 83% and a negative predictive value of 75%.

Example 3

The following example demonstrates a correlation between ASST and CD203cexpression.

The mean diameter of the serum autologous skin test was compared topercent CD203c expression. Results demonstrated a significantcorrelation between the size of the ASST and CD203c expression (R=0.5,p=0.02) (FIG. 5).

Example 4

The following example demonstrates that the mechanism of expression ofCD203c in CU patients is mediated by IgG.

In order to determine if the mechanism of CD203c expression was mediatedby an IgG antibody, 3 selected sera that induced basophil CD203cexpression were depleted of IgG using a protein G Sepharose. The meanpercent CD203c expression prior to IgG depletion was 30%±9%. The meanpercent CD203c expression post IgG depletion was 5%±1%, indicating thatin these sera CD203c expression may be triggered by an IgG antibody toFcεRIα. There was no change from pre and post IgG depletion with twocontrol sera 0.2±0.4 (pre) and −0.1±0.02 (post) (p=0.5).

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9. Boumiza R, Monneret G, Forissier M F, Savoye J, Gutowski M C, PowellW S, Bienveu J. Marked improvement of the basophil activation test bydetecting CD203c instead of CD63. Clin Exp Allergy 2002;33:259-65.

10. Binder M, Fierlbeck G, King T, Valent P, Burhing H J. Individualhymenoptera venom compounds induce upregulation of the basophilactivation marker ectonucleotide pyrophosphatase/phosphodiesterase 3(CD203c) in sensitized patients. Int Arch Allergy Immunol 2002;129:160-8.

11. Hauswirth A W, Natter S, Ghannadan M, Majlesi Y, Schernthaner G H,Sperr W R, Buhring H J, Valenta R, Valent P. Recombinant allergenspromote expression of CD203c on basophils in sensitized individuals. JAllergy Clin Immunol 2002; 127:299-307.

12. Vasgar K, Vonakis, B M, Viksman, A, Gibbons S., Jr. Saini, S.Evidence of in vivo basophil activation in chronic idiopathic urticaria.J Allergy Clin Immunol 2003;113:S257.

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14. Burhing H J, Seiffert M, Giesert C, Marxer A, Valent P, Kanz L, SanoK. The basophil activation marker defined by antibody 97A6 is identicalwith ecto-nucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3;CD203c). Blood 2001;97:3303-3305.

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Each reference cited herein is incorporated herein by reference in itsentirety.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1. A method to diagnose autoimmune chronic urticaria, comprising: a)contacting isolated cells with a test biological sample from anindividual being diagnosed for autoimmune chronic urticaria, wherein theisolated cells are capable of expressing CD203c and FcεRIα, andupregulate the expression of CD203c upon stimulation of the FcεRIα; andb) detecting expression of CD203c expression on the cells, whereinupregulation of CD203c on the cells in the presence of the testbiological sample, as compared to CD203c expression on the cells in theabsence of contact with the biological sample, or as compared to CD203cexpression on the cells when contacted with a negative control samplefrom an individual who does not have autoimmune chronic urticaria,indicates that the individual has autoimmune chronic urticaria.
 2. Themethod of claim 1, wherein the test biological sample is a fluid sample.3. The method of claim 1, wherein the test biological sample is serum orwhole blood from the individual.
 4. The method of claim 1, wherein theisolated cells are provided as a whole blood sample from a donor or asisolated peripheral blood cells from a donor.
 5. The method of claim 1,wherein the isolated cells are provided as substantially purified cellsfrom a donor.
 6. The method of claim 1, wherein the isolated cells areprovided as a cell line.
 7. The method of claim 1, wherein the isolatedcells are from a donor that does not have autoimmune chronic urticaria.8. The method of claim 1, wherein the isolated cells have been selectedas having high upregulation of CD203c expression after stimulation witha granulocyte activating agent and/or after stimulation of FcεRIα on thecells, as compared to isolated cells from a population of normalindividuals that do not have autoimmune chronic urticaria.
 9. The methodof claim 1, wherein the isolated cells are from a non-atopic donor. 10.The method of claim 1, wherein the isolated cells are not cultured withinterleukin-3 prior to the step of contacting.
 11. The method of claim1, wherein the isolated cells are selected from the group consisting of:basophils and mast cells.
 12. The method of Clam 1, wherein the step ofdetecting is performed by flow cytometry.
 13. The method of Clam 1,wherein the step of detecting is performed using a CD203c antibody orantigen-binding fragment thereof.
 14. The method of claim 13, whereinthe antibody is a monoclonal antibody.
 15. The method of claim 1,wherein the cells express CD13, and wherein the method further comprisesdetecting expression of CD13 by the cells, wherein increased expressionof CD13 in the presence of the test biological sample, as compared toexpression of CD13 by the cells in the absence of contact with thebiological sample, or as compared to CD13 expression on the cells whencontacted with a negative control sample from an individual who does nothave autoimmune chronic urticaria, further indicates that the individualhas autoimmune chronic urticaria.
 16. The method of claim 1, wherein thecells express CD164, and wherein the method further comprises detectingexpression of CD164 by the cells, wherein increased expression of CD164in the presence of the test biological sample, as compared to expressionof CD164 by the cells in the absence of contact with the biologicalsample, or as compared to CD164 expression on the cells when contactedwith a negative control sample from an individual who does not haveautoimmune chronic urticaria, further indicates that the individual hasautoimmune chronic urticaria.
 17. The method of claim 1, wherein thecells express CD63, and wherein the method further comprises detectingCD63 expression on the cells, wherein increased expression of CD63 inthe presence of the test biological sample, as compared to CD63expression on the cells in the absence of contact with the biologicalsample, or as compared to CD63 expression on the cells when contactedwith a negative control sample from an individual who does not haveautoimmune chronic urticaria, further indicates that the individual hasautoimmune chronic urticaria.
 18. The method of claim 1, furthercomprising testing the individual by histamine release assay, wherein apositive histamine release assay further indicates that the individualhas autoimmune chronic urticaria.
 19. The method of claim 1, furthercomprising testing the individual by autologous serum skin testing(ASST), wherein a positive ASST test further indicates that theindividual has autoimmune chronic urticaria.
 20. A kit for the diagnosisof autoimmune chronic urticaria, comprising: a) isolated cells that arecapable of expressing CD203c and FcεRI and that upregulate theexpression of CD203c upon stimulation of FcεRI; and b) a reagent for thedetection of CD203c expression on the cells.
 21. The kit of claim 20,wherein the reagent for detection of CD203c expression on the cells is aCD203c antibody or antigen-binding fragment thereof.
 22. The kit ofclaim 20, wherein the antibody is a monoclonal antibody.
 23. The kit ofclaim 20, further comprising a reagent selected from the groupconsisting of a positive control reagent and a negative control reagent.24. The kit of claim 23, wherein the positive control reagent isselected from the group consisting of fMLP and anti-FcεRIα.
 25. The kitof claim 23, wherein the negative control reagent comprises a buffer.26. The kit of claim 20, wherein the isolated cells are basophils.
 27. Amethod to identify a compound that inhibits autoimmune chronicurticaria, comprising: a) contacting isolated cells that are capable ofexpressing CD203c and FcεRIα, and that upregulate the expression ofCD203c upon stimulation of the FcεRIα, with an agent that stimulates theexpression of CD203c by the cells; b) contacting the isolated cells witha putative regulatory compound; and c) detecting expression of CD203cexpression on the cells, wherein prevention or inhibition ofupregulation of CD203c on the cells in the presence of the putativeregulatory compound, as compared to the upregulation of CD203cexpression on the cells in the absence of contact with the putativeregulatory compound, indicates that the putative regulatory compound isa candidate for inhibition of autoimmune chronic urticaria.
 28. A methodto treat autoimmune chronic urticaria, comprising administering to theindividual a pharmaceutical composition that inhibits the expression orbiological activity of CD203c.